I would like to hear about your best practices advice for immunostaining of organoids.

Answer

Organoids can either be recovered from the hydrogel/extracellular matrix or they can be stained in the matrix itself depending on what matrix and concentration is used. For recovering organoids from Corning Matrigel matrix, the culture can be treated with Corning Cell Recovery Solution (not Dispase as it will make single cell suspension, unless this is the desired result) to release organoids and then proceed with fixing, permeabilizing and staining the organoids. Another option is to stain the organoids directly in Matrigel matrix. This method can give higher background, so blocking/washing will need to be optimized for optimum signal: noise ratio. The use of phenol red-free Matrigel is recommended to reduce background during immunostaining. Often, permeabilization and staining concentrations will need to be higher and incubation steps will be longer in order to penetrate through the entire organoid. If embedding organoids prior to processing, Corning has a protocol available that guides you through the spheroid processing and embedding for histology guidelines (CLS-AN-431). Visikol, is a clearing agent that allows better visualization of 3D structures that have been immuno-labelled. Requirement for antigen retrieval and subsequent staining protocols for embedded organized will have to be optimized for target antigen. Please see some references below:

Nguyen-Ngoc, Kim-Vy, et al. “3D culture assays of murine mammary branching morphogenesis and epithelial invasion.” Tissue Morphogenesis: Methods and Protocols (2015): 135-162.

Lee, Genee Y., et al. “Three-dimensional culture models of normal and malignant breast epithelial cells.” Nature methods 4.4 (2007): 359-365.